Orally bioavailable lipid-based constructs

ABSTRACT

The present invention is embodied by a composition capable inducing weight loss in a patient in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part U.S. application Ser. No.12/732,952, filed Mar. 26, 2010, which is a continuation in part of PCTapplication PCT/US08/77990, filed on Sep. 26, 2008, which in turn claimspriority to U.S. patent application Ser. No. 11/904,937, filed on Sep.28, 2007, each of which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

One of the most preferred ways to deliver a pharmaceutical to a subjectis in an oral formulation. However, oral formulations of manypharmaceutical compounds are often unavailable due to thepharmaceutical's incompatibility with the harsh environment of thedigestive tract. This is particularly true for pharmaceutical compoundssuch as peptides, proteins, certain small molecules, and nucleic acids.

Another issue plaguing oral delivery is the quantity of medication thatmust be both orally administered to affect the desired outcome in apatient. For example, poor bioavailability due to a bad solubilityprofile can mean that even though a certain medication tolerates thedigestive milieu, it cannot be given orally in any meaningful way. Itmay, for example, need to be given in a substantially larger doses thanwould be required if given intravenously, or via another route ofadministration.

D-biotin is an example of a compound that, while susceptible to oraldelivery, has a poor solubility profile. As a result, the amount ofmaterial that must be given to ensure activity in general, and inparticular at the liver, is substantially larger than preferred. Thus,what is needed in the field of biotin delivery, is a composition capableof affecting efficient oral delivery of biotin and a biotin-derivedcompounds. The present invention meets these needs.

BRIEF SUMMARY OF THE INVENTION

The present invention includes compositions that facilitate and/orenable efficient oral absorption of biotin or biotin-derived compoundsthat are not presently efficiently processed by the body when givenalone. The various compositions of the invention described herein havebeen found to improve biotin bioavailability by substantially increasingsolubility. In addition, the compositions described herein have beensurprisingly found to affect weight loss in patients in need thereof ata dose that is substantially reduced from what was previously known inthe prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofpreferred embodiments of the invention, will be better understood whenread in conjunction with the appended drawings. For the purpose ofillustrating the invention, there are shown in the drawings embodimentswhich are presently preferred. It should be understood, however, thatthe invention is not limited to the precise arrangements andinstrumentalities shown.

FIG. 1 is a schematic representation of a composition of the invention.

FIG. 2 is a graph depicting the counts of ¹⁴C radio-labeled phospholipidfound in the femoral and portal veins 15 and 30 minutes post injectingradio-labeled composition into the duodenum of a fasted and anesthetized230 gram rat.

FIG. 3 is a bar graph depicting the distribution of ¹⁴C radio-labeledphospholipid amongst the blood, liver, and spleen in the rats of FIG. 2,post-sacrifice.

FIG. 4 is a graph depicting the absorption of radio-labeled compositionfrom drinking water at 15, 30, and 45 minutes post-dosing.

FIG. 5 is a bar graph depicting the distribution of the labeledcomposition amongst the blood, liver, and spleen in the rats of FIG. 4,post-sacrifice.

FIG. 6 is a graph depicting the efficacy of orally administered insulinin the form of a composition of the invention.

FIG. 7 is a bar graph depicting the efficacy of a composition of theinvention (at low dosages), in converting a type 2 diabetic dog fromhepatic glucose output to uptake during a portal glucose load.

FIG. 8 is a plot of blood calcium levels after the administration ofcalcitonin associated with a non-targeted composition of the invention.

FIG. 9 is a graph of the size distribution of the constituent members ofa composition of the invention.

FIG. 10 is a graph of the efficacy of a composition of the inventioncomprising a biotin targeting agent and insulin at reducing the effectsof type 2 diabetes in humans.

FIG. 11 is a chromatogram of a composition of the invention showing theefficacy of insulin loading.

FIG. 12 is a graph depicting the efficacy of oral delivery of IgGantibodies covalently linked to a composition of the invention versusoral absorption of non-associated (free) IgG antibodies.

FIG. 13 is a graph depicting the effect of oral administration ofthyroxine associated with a composition of the invention on serumcholesterol and triglycerides (“TG”) in mice.

FIG. 14 is a graph depicting the effect of oral administration ofinterferon associated with a composition of the invention on reducingviral load in humans suffering from hepatitis-C.

FIG. 15 is a bar graph depicting the effect of various amounts of acomposition of the invention on fasting blood glucose after an 18 weekdosing regimen.

FIG. 16 is a bar graph depicting the effect of various amounts of acomposition of the invention on HbA1c in Type 2 diabetics after an 18week dosing regimen.

FIG. 17 is a bar graph depicting weight loss in a total population ofpatients dosed with various amounts of a composition of the inventionafter an 18 week dosing regimen.

FIG. 18 is a bar graph depicting weight loss in a sub-population ofpatients dosed with various amounts of a composition of the inventionafter an 18 week dosing regimen.

FIG. 19 is a graph of the 7 point blood glucose index of the “Intent ToTreat” population as of the last observation carried forward (“LOCF”)with a composition of the invention.

FIG. 20 is a line graph showing the expected weight loss over a 1 yearperiod in a population of patients dosed with various amounts of acomposition of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes compositions that facilitate and/orenable efficient oral absorption of biotin or biotin-derived compoundsthat are not presently efficiently processed by the body when givenalone. The various compositions of the invention described herein havebeen found to improve biotin bioavailability by substantially increasingsolubility. In addition, the compositions described herein have beensurprisingly found to affect weight loss in patients in need thereof ata dose that is substantially reduced from what was previously known inthe prior art.

In one embodiment, a composition of the invention comprises variouslipid components (which can themselves be members of a liposome orliposome fragment), an optional targeting agent, D-biotin, and gelatin.In this embodiment, the composition associates with D-biotin, andchaperones it through the lumen of the gut into the portal blood flowand finally on to the systemic circulation. In other embodiments, thecomposition of the invention comprises various lipid components, abiotin-derived targeting agent, and gelatin.

In some embodiments, a composition of the invention can comprise gelatinand about 56 mole percent 1,2 distearoyl-sn-glycero-3-phosphocholine,about 21 mole percent dihexadecyl phosphate, about 15 mole percentcholesterol, and about 8 mole percent of a biotin-derived targetingagent.

In some embodiments, the biotin-derived targeting agent is selected fromthe group consisting of N-hydroxysuccinimide (NHS) biotin;sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin;sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin;sulfo-N-hydroxysuccinimide-S—S-biotin; biotin-BMCC; biotin-HPDP;iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytinhydrazide; biotin cadaverine; carboxybiotin; photobiotin; ρ-aminobenzoylbiocytin trifluoroacetate; ρ-diazobenzoyl biocytin; biotin DHPE;biotin-X-DHPE; 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide; biotmethylenediamine;biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE;biotin-XX, SSE; biotin-X-cadaverine; α-(t-BOC)biocytin;N-(biotinyl)-N′-(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE;biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-1-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol)amine; (+)biotin 4-amidobenzoic acid sodium salt; Biotin2-N-acetylamino-2-deoxy-β-D-glucopyranoside;Biotin-α-D-N-acetylneuraminide; Biotin-α-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-α-D-mannopyranoside; biotin6-O-phospho-α-D-mannopyranoside; and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl),iminobiotin derivatives of the aforementioned compounds, and mixturesthereof.

In particular embodiments, the biotin-derived targeting agent isD-biotin, biotin DHPE, or biotin-X-DHPE.

The present invention also provides a method for making an orallybioavailable composition comprising gelatin and about 56 mole percent1,2 distearoyl-sn-glycero-3-phosphocholine, about 21 mole percentdihexadecyl phosphate, about 15 mole percent cholesterol, and about 8mole percent of a biotin-derived targeting agent. The method cancomprise the steps of mixing said 1,2distearoyl-sn-glycero-3-phosphocholine, dihexadecyl phosphate,cholesterol and said at least one a biotin-derived targeting agent inaqueous media to form a mixture; adding said mixture to gelatin to forma gelatin-associated mixture; and drying said gelatin-associatedmixture.

In particular embodiments of the method described herein, thebiotin-derived targeting agent is selected from the group consisting ofD-biotin, biotin DHPE, and biotin-X-DHPE.

The present invention also provides a method of affecting weight loss ina patient, said method comprising administering to said patient aneffective amount of an orally bioavailable composition comprisinggelatin and about 56 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, about 21 mole percentdihexadecyl phosphate, about 15 mole percent cholesterol, and about 8mole percent of a biotin-derived targeting agent.

In certain embodiments of the method of affecting weight loss, thebiotin-derived targeting agent is D-biotin, biotin DHPE, orbiotin-X-DHPE.

In another embodiment of the invention, the composition of the inventionfurther comprises D-biotin.

In an embodiment, a method of the invention includes co-administering atleast one therapeutic agent useful for inducing weight loss

In some embodiments, the at least one therapeutic agent useful forinducing weight loss is orlistat, sibutramine, phendimetrazine tartrate,methamphetamine, IONAMIN™, phentermine, fenfluramine, dexfenfluramine,chitosan, chromium picolinate, conjugated linoleic acid, green teaextract, guar gum, hoodia, a combination of topiramate and phentermine,a combination of bupropion and zonisamide, a combination of bupropionand naltrexone, a combination of phentermine and fluoxetine, acombination of phentermine and sertraline, a combination of phentermineand citalopram, a combination of phentermine and escitalopram, or acombination of phentermine and trazodone.

In one embodiment, the invention is a food additive, dietary supplement,or beverage additive comprising gelatin and about 56 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, about 21 mole percentdihexadecyl phosphate, about 15 mole percent cholesterol, and about 8mole percent of a targeting agent.

In some embodiments, the biotin-derived targeting agent is selected fromthe group consisting of N-hydroxysuccinimide (NHS) biotin;sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin;sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin;sulfo-N-hydroxysuccinimide-S—S-biotin; biotin-BMCC; biotin-HPDP;iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytinhydrazide; biotin cadaverine; carboxybiotin; photobiotin; ρ-aminobenzoylbiocytin trifluoroacetate; ρ-diazobenzoyl biocytin; biotin DHPE;biotin-X-DHPE; 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide; biotmethylenediamine;biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE;biotin-XX, SSE; biotin-X-cadaverine; α-(t-BOC)biocytin;N-(biotinyl)-N′-(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE;biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-1-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol)amine; (+)biotin 4-amidobenzoic acid sodium salt; Biotin2-N-acetylamino-2-deoxy-β-D-glucopyranoside;Biotin-α-D-N-acetylneuraminide; Biotin-α-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-α-D-mannopyranoside; biotin6-O-phospho-α-D-mannopyranoside; and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl),iminobiotin derivatives of the aforementioned compounds, and mixturesthereof.

In other embodiments, the biotin-derived targeting agent is D-biotin,biotin DHPE, or biotin-X-DHPE.

The present invention further provides a method of maintaining theweight of a patient in need thereof, comprising administering a compoundcomprising gelatin and about 56 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, about 21 mole percentdihexadecyl phosphate, about 15 mole percent cholesterol, and about 8mole percent of a targeting agent.

In some embodiments, the biotin-derived targeting agent is selected fromthe group consisting of N-hydroxysuccinimide (NHS) biotin;sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin;sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin;sulfo-N-hydroxysuccinimide-S—S-biotin; biotin-BMCC; biotin-HPDP;iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytinhydrazide; biotin cadaverine; carboxybiotin; photobiotin; ρ-aminobenzoylbiocytin trifluoroacetate; ρ-diazobenzoyl biocytin; biotin DHPE;biotin-X-DHPE; 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide; biotmethylenediamine;biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE;biotin-XX, SSE; biotin-X-cadaverine; α-(t-BOC)biocytin;N-(biotinyl)-N′-(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE;biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-1-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol)amine; (+)biotin 4-amidobenzoic acid sodium salt; Biotin2-N-acetylamino-2-deoxy-β-D-glucopyranoside;Biotin-α-D-N-acetylneuraminide; Biotin-α-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-α-D-mannopyranoside; biotin6-O-phospho-α-D-mannopyranoside; and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl),iminobiotin derivatives of the aforementioned compounds, and mixturesthereof.

In particular embodiments, the biotin-derived targeting agent isD-biotin, biotin DHPE, or biotin-X-DHPE.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which the invention belongs. Generally, thenomenclature used herein and the laboratory procedures in organicchemistry and protein chemistry are those well known and commonlyemployed in the art.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, amino acids are represented by the full name thereof, bythe three-letter code as well as the one-letter code correspondingthereto, as indicated in the following table:

3 Letter 1-Letter Full Name Code Code Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic Acid Asp D Cysteine Cys C Cystine Cys-Cys C-CGlutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

The term “lower”, when used in reference to a chemical structure,describes a group containing from 1 to 6 carbon atoms.

The term “alkyl”, by itself or as part of another substituent means,unless otherwise stated, a straight, branched or cyclic hydrocarbonhaving the number of carbon atoms designated (i.e. C₁-C₆ means one tosix carbons). Examples include: methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, pentyl, neopentyl, hexyl, cyclohexyl andcyclopropylmethyl. Most preferred is (C₁-C₃) alkyl, particularly ethyl,methyl and isopropyl.

The term “alkylene”, by itself or as part of another substituent means,unless otherwise stated, a straight, branched or cyclic chainhydrocarbon having two substitution sites, e.g., methylene (—CH₂—),ethylene (—CH₂CH₂—), isopropylene (—C(CH₃)═CH—), etc.

The term “aryl”, employed alone or in combination with other terms,means, unless otherwise stated, a carbocyclic structure, with or withoutsaturation, containing one or more rings (typically one, two or threerings) wherein said rings may be attached together in a pendant manner,such as a biphenyl, or may be fused, such as naphthalene. Examplesinclude phenyl, anthracyl, and naphthyl. The structure may be optionallysubstituted with one or more substituents, independently selected fromhalogen; (C₁-C₆)alkyl; (C₁-C₆)alkenyl; (C₁-C₆)alkoxy; OH; NO₂; C≡N;C(═O)O(C₁-C₃)alkyl; (C₂-C₆)alkylene-OR²; phosphonato; NR² ₂;NHC(═O)(C₁-C₆)alkyl; sulfamyl; carbamyl; OC(═O)(C₁-C₃)alkyl;O(C₂-C₆)alkylene-N((C₁-C₆)alkyl)₂; and (C₁-C₃)perfluoroalkyl.

The term “arylloweralkyl” means a functional group wherein an aryl groupis attached to a lower alkylene group, e.g., —CH₂CH₂-phenyl.

The term “alkoxy” employed alone or in combination with other termsmeans, unless otherwise stated, an alkyl group or an alkyl groupcontaining a substituent such as a hydroxyl group, having the designatednumber of carbon atoms connected to the rest of the molecule via anoxygen atom, such as, for example, —OCH(OH)—, —OCH₂OH, methoxy (—OCH₃),ethoxy (—OCH₂CH₃), 1-propoxy (—OCH₂CH₂CH₃), 2-propoxy(isopropoxy),butoxy (—OCH₂CH₂CH₂CH₃), pentoxy (—OCH₂CH₂CH₂CH₂CH₃), and the higherhomologs and isomers.

The term “acyl” means a functional group of the general formula—C(═O)—R, wherein —R is hydrogen, alkyl, amino or alkoxy. Examplesinclude acetyl (—C(═O)CH₃), propionyl (—C(═O)CH₂CH₃), benzoyl(—C(═O)C₆H₅), phenylacetyl (C(═O)CH₂C₆H₅), carboethoxy (—CO₂CH₂CH₃), anddimethylcarbamoyl (C(═O)N(CH₃)₂).

The terms “halo” or “halogen” by themselves or as part of anothersubstituent mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom.

The term “heterocycle” or “heterocyclyl” or “heterocyclic” by itself oras part of another substituent means, unless otherwise stated, asaturated or unsaturated, stable, mono or multicyclic ring systemcomprising carbon atoms and at least one heteroatom selected from thegroup comprising N, O, and S, and wherein the nitrogen and sulfurheteroatoms may be optionally oxidized, and the nitrogen atom may beoptionally quaternized. Examples include pyridine, pyrrole, imidazole,benzimidazole, phthalein, pyridenyl, pyranyl, furanyl, thiazole,thiophene, oxazole, pyrazole, 3-pyrroline, pyrrolidene, pyrimidine,purine, quinoline, isoquinoline, carbazole, etc. Where substitution willresult in a stable compounds, the structure may be optionallysubstituted with one or more substituents, independently selected fromhalogen; (C₁-C₆)alkyl; (C₁-C₆)alkenyl; (C₁-C₆)alkoxy; OH; NO₂; C≡N;C(═O)O(C₁-C₃)alkyl; (C₂-C₆)alkylene-OR²; phosphonato; NR² ₂;NHC(═O)(C₁-C₆)alkyl; sulfamyl; carbamyl; OC(═O)(C₁-C₃)alkyl;O(C₂-C₆)alkylene-N((C₁-C₆)alkyl)₂; and (C₁-C₃)perfluoroalkyl.

The term “amphipathic lipid” means a lipid molecule having a polar endand a non-polar end.

A “complexing agent” is a compound capable of forming a water insolublecoordination complex with a metal, e.g. a salt of chromium, zirconium,etc., that is substantially insoluble in water and soluble in organicsolvents.

“Aqueous media” means media comprising water or media comprising watercontaining at least one buffer or salt.

The terms “associated,” or “associated with” when used in reference to acomposition or constituent of a composition of the invention, means thatthe referenced material is incorporated (or intercalated) into, or onthe surface of, or within a composition or a constituent of acomposition of the present invention.

The term “insulin” refers to natural or recombinant forms of insulin,synthetic insulin, and derivatives of the aforementioned insulins.Examples of insulin include, but are not limited to insulin lispro,insulin aspart, regular insulin, insulin glargine, insulin zinc, humaninsulin zinc extended, isophane insulin, human buffered regular insulin,insulin glulisine, recombinant human regular insulin, ultralenteinsulin, humulin, NPH insulin, Levemir, Novolog, and recombinant humaninsulin isophane. Also included are animal insulins, such as bovine orporcine insulin.

The terms “glargine” and “glargine insulin” both refer to a recombinanthuman insulin analog which differs from human insulin in that the aminoacid asparagine at position A21 is replaced by glycine and two argininesare added to the C-terminus of the B-chain. Chemically, it is21A-Gly-30Ba-L-Arg-30Bb-L-Arg-human insulin and has the empiricalformula C₂₆₇H₄₀₄N₇₂O₇₈S₆ and a molecular weight of 6063.

The term “recombinant human insulin isophane” refers to a human insulinthat has been treated with protamine.

The term “bioavailability” refers to a measurement of the rate andextent that a pharmaceutical agent, such as, but not limited to,insulin, reaches the systemic circulation and is available at its siteof action.

As used herein, to “treat” means reducing the frequency with whichsymptoms of a disease, disorder, or adverse condition, and the like, areexperienced by a patient.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject.

The term “lipid” or “lipids” means an organic compound characterized byits preference for non-polar aprotic organic solvents. A lipid may ormay not possess an alkyl tail. Lipids according to the present inventioninclude, but are not limited to, the class of compounds known in the artas phospholipids, cholesterols, and dialkyl phosphates.

As used herein, “cholesterol” means the compound and all derivatives andanalogs of the compound:

As used herein, “particle” comprises an agglomeration of multiple unitsof one or more lipids.

As used herein, “thyroxine” refers to the compound:

wherein the amino group may be in either the “D” or “L” configuration.

As used herein, “co-administration” or “co-administering” or“co-administered” as well as variations thereof, mean administering acomposition of the invention before, during, or after the administrationof one or more additional therapeutic agents wherein the one or moreadditional therapeutic agents is/are not associated with the compositionof the invention. Co-administration may take place via the same ordifferent routes of administration. Co-administration may be concurrent,sequential, or spaced at specific time intervals. Co-administration neednot, however, take place within a set time period. As such, and by wayof example only, administration of a composition of the invention at anytime before or after the administration of one or more additionaltherapeutic agents constitutes co-administration so long as either acomposition of the invention or the one or more additional therapeutics(whichever is administered first) is still present in the patient at thetime of co-administration. In certain embodiments, though, the firstadministered compound need not be present in the patient at the time ofco-administration.

As used herein, “interferon” refers to all forms of interferon,including, but not limited to, interferon-α, interferon-beta,interferon-gamma, as well as sub-units thereof.

DESCRIPTION

A composition of the present invention is comprised of gelatin and oneor more constituents wherein said constituents include liposomes,liposome fragments, and lipid particles.

Traditionally, liposome, liposome fragments, and lipid particlescomprised of amphipathic materials have been limited to a lower sizedistribution of about 40 nanometers. This limit was believed to be afunction of the collective sizes of the constituent lipids(phospholipids, cholesterols, dialkylphosphates, etc.) that constitutedthe membrane structure.

The constituents of a composition of the present invention, however,demonstrate heretofore unobserved dynamic sizing and size elasticity.Specifically, constituents of the compositions of the present invention,exist in a dynamic equilibrium in aqueous media wherein theconstituents, on average, fluctuate in size from about 6 nanometers toabout 60 nanometers in diameter. At any given time, anywhere from about5% to about 50% of the constituents exhibit an average diameter of about20 nanometers or less. Due to the nearly constant fluctuations in sizes,the constituents of the compositions of the present invention cannot bephysically separated by traditional fractionating means to form discretepopulations of differently sized structures. The constituents of acomposition of the invention may be, but are not limited to, a liposome,a liposome fragment, and a lipid particle. The composition of theinvention can further optionally include a targeting agent.

Lipids

A constituent of a composition of the present invention comprises one ormore lipid components and an optional targeting agent. An embodimentcomprising a single unit or multiple units of a single lipid componentis referred to herein as a “lipid particle.” An embodiment comprisingtwo or more different lipid components and an optional targeting agentis classified as a liposome or liposome fragment, depending upon thenature of the resulting structure.

Lipid components of the present invention are selected from the groupconsisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine, cholesterol, cholesterololeate, dihexadecyl phosphate, 1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt),triethylammonium 2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethylphosphate, MPB-PE and derivatives thereof. Representative structures arepresented in Table 1.

TABLE 1 Common Name Chemical Name Structure 1,2-distearoyl-sn-glycero-3- phosphocholine 2,3- bis(stearoyloxy)propyl2-(trimethylammonio) ethyl phosphate

1,2-dipalmitoyl- sn-glycero-3- phosphocholine 2,3-bis(palmitoyloxy)propyl 2-(trimethylammonio) ethyl phosphate

1,2-dimyristoyl- sn-glycero-3- phosphocholine 2,3-bis (tetradecanoyloxy)propyl 2- (trimethylammonio) ethyl phosphate

Cholesterol 10,13-dimethyl-17- (6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13, 14,15,16,17- tetradecahydro-1H-cyclopenta[a]phenan- thren-3-ol

MPB-PE

By way of non-limiting examples, the constituents of a composition ofthe present invention may be formed from lipid components mixed inaccordance with the following: approximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, approximately 22 mole percentdihexadecyl phosphate, and approximately 16 mole percent cholesterol. Inembodiments wherein a constituent incorporates a targeting agent, theabove noted mixture may further include from about 1 to about 2 molepercent of at least one targeting agent, with the amounts of other lipidcomponents reduced to maintain the ratio of components set forth above.

In another embodiment, a composition of the present invention may beformed from lipid components mixed in accordance with the following:approximately 68 mole percent 1,2dipalmitoyl-sn-glycero-3-phosphocholine, approximately 18 mole percentdihexadecyl phosphate, approximately 9 mole percent cholesterol, andapproximately 3 percent MPB-PE. In embodiments wherein a constituentincorporates a targeting agent, the above noted mixture may furtherinclude from about 1 to about 2 mole percent of at least one targetingagent, with the amounts of other lipid components reduced to maintainthe ratio of components set forth above.

Preparation

Generally, the constituents of a composition of the present inventionare formed when at least one lipid component and optional targetingagent are homogenized in an aqueous media via microfluidization or otherprocess involving cavitation.

In an embodiment of the invention, the lipid component(s) and optionaltargeting agent(s) may be homogenized in 18 mM phosphate buffer at a pHof about 6.0 to a pH of about 8.0. Lipid component concentration in thephosphate buffer may range from about 10 to about 200 mg/ml and any andall whole and partial integers therebetween. In one embodiment, thelipid component concentration is about 30 to about 150 mg/ml. In morepreferred embodiment, the lipid component concentration is about 15 toabout 50 mg/ml. In a most preferred embodiment, the lipid componentconcentration is about 28-30 mg/ml.

Homogenization of the aqueous media, lipid component(s), and optionaltargeting agent may be accomplished via treatment in a device suitablefor homogenization. Examples of suitable devices include, but are notlimited to, a Polytron® System PT 6100, an M-110-EH microfluidizer, anultrasonic sonicator, a high pressure membrane filtration apparatus, anda homogenizer extruder.

In instances where a microfluidizer is used, the microfluidizer ispreferably operated at a temperature that is greater than the highesttransition temperature of a lipid component and most preferably at atemperature greater than about 75° C. Thus, the elevated temperatureallows any acyl and alkyl chains present in the lipid component(s) tomove fluidly as well as conform to and associate with neighboringhydrocarbon moieties. These non-covalent associations directly result inthe formation of a constituent of a composition of the presentinvention.

For the microfluidization process, up to about five independent passesare required at 9000 psig in order to achieve dynamic constituent sizingwith some constituents possessing radii of less than 20 nanometers.Constituent analysis data generated by a Coulter N-4 Plus Sub-MicronParticle Size Analyzer is shown in FIG. 9 and represents 10 repeatedsize analyses on the same sample as it remained stationary in theCoulter N-4 Plus Sub-Micron Particle Size Analyzer. This datademonstrates the dynamic nature of constituent sizing and the fluidnature of the interactions between the constituents of the compositionof the present invention in aqueous media.

After microfluidization, the resulting constituents may be sterilefiltered through a 0.8 micron to 0.2 micron gang Supor™ membrane.

During the process of sub-micron particle formation, hydrogen bonding,ionic bonding, van der Waal's interactions, dipolar interactions,ion-dipole interactions and hydrophobic associations dictate the mannerin which the constituents of a composition of the present inventionassemble. While not wishing to be bound by any one particular theory, itis believed that the interaction of all of these forces, to varyingextents, under the conditions noted above, lead to the dynamically sizedconstituents of the present invention.

Incorporation of a Targeting Agent

In certain embodiments, a constituent of the present invention mayoptionally comprise a targeting agent. Targeting agents alter aconstituent's bio-distribution and further enhance the efficacy of anassociated therapeutic agent. For example, a constituent of acomposition of the present invention may incorporate one or moretargeting agents that act to target the constituent to a specificcellular or extracellular receptor. Alternatively, by way of anon-limiting example, the targeting agent may mask the constituent fromreticuloendothelial (macrophage) recognition.

In one embodiment, a targeting agent facilitates delivery of thecomposition to the liver and encompasses a class of molecules referredto as “hepatocyte target molecule” (HTM). HTM examples include biotinderived targeting agents such as1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) and metalderived targeting agents such aspoly[Cr-bis(N-2,6-diisopropylphenylcarbamoylmethyl iminodiacetic acid)].Metal-derived targeting agents and biotin derived targeting agents arediscussed below and are fully described in U.S. Pat. Nos. 7,169,410 and4,603,044; PCT application PCT/US06/19119; and U.S. patent applicationSer. Nos. 11/384,728, and 11/384,659. Additional examples ofbiotin-derived targeting agents are disclosed in Table 2.

When the targeting agent comprises biotin, iminobiotin, carboxybiotin,biocytin, or iminobiocytin, the biotin, iminobiotin, carboxybiotin,biocytin, or iminobiocytin molecules may be bound via an amide bond tothe nitrogen of a phospholipid molecule such as1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine. The compounds maylikewise be bound to a molecule such as cholesterol through an esterlinkage. In the case of biocytin and iminobiocytin, the compounds may bebound to benzoyl thioacetyl triglycine via an amide bond between theterminal nitrogen of iminiobiocytin and the terminal carbonyl of benzoylthioacetyl triglycine. Alternative bond connectivities to thosedescribed above are possible and considered to be within the scope ofthe present invention.

TABLE 2 1 N-hydroxysuccinimide (NHS) biotin 2,5-dioxopyrrolidin-1-yl 5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl) pentanoate

2 sulfo-NHS-biotin sodium 2,5-dioxo-3- (trioxidanylthio)pyrrolidin-1-yl5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4-yl) pentanoate

3 N-hydroxysuccinimide long chain biotin 2,5-dioxopyrrolidin-1-yl 6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoate

4 sulfo-N-hydroxysuccinimide long chain biotin sodium 2,5-dioxo-3-(trioxidanylthio)pyrrolidin-1-yl 6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl)pentanamido) hexanoate

5 D-biotin 5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4-yl)pentanoic acid

6 Biocytin 2-amino-6-(5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido) hexanoic acid

7 sulfo-N-hydroxysuccinimide-S- S-biotin sodium 2,5-dioxo-3-(trioxidanylthio)pyrrolidin-1-yl 3-((2-(4-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl)butylamino)ethyl)disulfanyl)propanoate

8 biotin-BMCC 4-((2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)methyl)-N-(4-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)butyl) cyclohexanecarboxamide

9 biotin-HPDP 5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)- N-(6-(3-(pyridin-2-yldisulfanyl)propanamido)hexyl)pentanamide

10 iodoacetyl-LC-biotin N-(6-(2-iodoacetamido)hexyl)-5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4- yl)pentanamide

11 biotin-hydrazide 5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4- yl)pentanehydrazide

12 biotin-LC-hydrazide N-(6-hydrazinyl-6-oxohexyl)-5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl) pentanamide

13 biocytin hydrazide N-(5-amino-6-hydrazinyl-6-oxohexyl)-5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide

14 biotin cadaverine N-(5-aminopentyl)-5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanamide

15 Carboxybiotin (3aS,6aR)-4-(4-carboxybutyl)-2-oxohexahydro-1H-thieno[3,4- d]imidazole-1-carboxylic acid

16 Photobiotin N-(3-((3-(4-azido-2- nitrophenylamino)propyl)(methyl)amino)propyl)-5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide

17 ρ-aminobenzoyl biocytin trifluoroacetate 2-(4-aminobenzamido)-6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoic acid 2,2,2-trifluoroacetate

18 ρ-diazobenzoyl biocytin 4-(1-carboxy-5-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanamido)pentylcarbamoyl) benzenediazonium chloride

19 biotin DHPE G⁺= Li⁺, Na⁺, K⁺, (Et₃NH)⁺ 2,3-diacetoxypropyl 2-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate

20 biotin-X-DHPE G⁺= Li⁺, Na⁺, K⁺, (Et₃NH)⁺ 2,3-diacetoxypropyl 2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate

21 12-((biotinyl)amino)dodecanoic acid 12-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl)pentanamido) dodecanoic acid

22 12-((biotinyl)amino)dodecanoic acid succinimidyl ester2,5-dioxopyrrolidin-1-yl 12-(5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanamido)dodecanoate

23 S-biotinyl homocysteine 4-mercapto-2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanamido) butanoic acid

24 biocytin-X 2-amino-6-(6-(5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido) hexanamido)hexanoic acid

25 biocytin x-hydrazide N-(5-amino-6-hydrazinyl-6-oxohexyl)-6-(5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido) hexanamide

26 Biotinethylenediamine N-(2-aminoethyl)-5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanamide

27 biotin-X 6-(5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido) hexanoic acid

28 biotin-X-ethylenediamine N-(2-aminoethyl)-6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamide

29 biotin-XX hydrazide N-(6-hydrazinyl-6-oxohexyl)-6-(5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamide

30 biotin-XX-SE 2,5-dioxopyrrolidin-1-yl 6-(6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido) hexanoate

31 biotin-XX, SSE sodium 2,5-dioxo-1-(6-(6-(5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hex- anoyloxy)pyrrolidine-3-sulfonate

32 biotin-X-cadaverine 5-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl)pentanamido)hexanamido)pentan-1-aminium 2,2,2-trifluoroacetate

33 α-(t-BOC)biocytin 2-(tert-butoxycarbonylamino)-6-(5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoic acid

34 N-(biotinyl)-N′- (iodoacetyl)ethylenediamineN-(2-(2-iodoacetamido)ethyl)-5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanamide

35 DNP-X-biocytin-X-SE 2,5-dioxopyrrolidin-1-yl 2-(6-(6-(2,4-dinitrophenylamino) hexanamido)hexanamido)-6-(6-(5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexamido) hexanoate

36 biotin-X-hydrazide N-(6-hydrazinyl-6-oxohexyl)-5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl) pentanamide

37 norbiotinamine hydrochloride 4-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) butan-1-aminium chloride

38 3-(N-maleimidylpropionyl) biocytin 2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-6-(5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanamido)hexanoic acid

39 ARP; N′-(2-(aminooxy)acetyl)-5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanehydrazide

40 biotin-1-sulfoxide 5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanoic acid sulfoxide

41 biotin methyl ester methyl 5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d] imidazol-4-yl)pentanoate

42 biotin-maleimide 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N′-(5-((3aS,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl) hexanehydrazide

43 Biotin-poly(ethyleneglycol) amine aminomethyl polyethylene 5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl) pentanoate

44 (+) biotin 4-amidobenzoic acid sodium salt sodium 4-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno [3,4-d]imidazol-4-yl) pentanamido)benzoate

45 Biotin 2-N-acetylamino-2- deoxy-β-D-glucopyranoside((2R,5S)-3-acetamido-4,5- dihydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyltetrahydro- 2H-pyran-2-yl)methyl 5-((3aS,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl) pentanoate

46 Biotin-α-D-N-acetylneuraminide (2S,5R)-5-acetamido-4-hydroxy-3,3,4,5,6-pentamethyl-2-((5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanoyloxy)methyl)-6-(1,2,3-trihydroxypropyl)tetrahydro- 2H-pyran-2-carboxylic acid

47 Biotin-α-L-fucoside ((2R,5S)-3,4,5-trihydroxy- 2,3,4,5,6,6-hexamethyltetrahydro-2H-pyran- 2-yl)methyl 5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)pentanoate

48 Biotin lacto-N-bioside See end of table for name

49 Biotin-Lewis-A trisaccharide See end of table for name

50 Biotin-Lewis-Y tetrasaccharide See end of table for name

51 Biotin-α-D-mannopyranoside ((1R,4R)-2,3,4-trihydroxy-5-(hydroxymethyl)-1,2,3,4,5- pentamethylcyclohexyl)methyl5-((3aS,6aR)-2-oxohexahydro- 1H-thieno[3,4-d]imidazol-4-yl) pentanoate

52 biotin 6-O-phospho-α-D- mannopyranoside ((2R,5S)-3,4,5-trihydroxy-2,3,4,5,6-pentamethyl-6- (phosphonooxymethyl)tetra-hydro-2H-pyran-2-yl)methyl 5- ((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) pentanoate

Names of Compounds 48-50:

-   48.    ((2R,5S)-3-acetamido-5-hydroxy-6-(hydroxymethyl)-2,3,4,6-tetramethyl-4-((((2S,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyltetrahydro-2H-pyran-2-yl)methoxy)methyl)tetrahydro-2H-pyran-2-yl)methyl    5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate    ((2R,5S)-3-acetamido-5-hydroxy-6-(hydroxymethyl)-2,3,4,6-tetramethyl-4-((((2S,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyltetrahydro-2H-pyran-2-yl)methoxy)methyl)tetrahydro-2H-pyran-2-yl)methyl    5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate-   49.    (2R,3R,5S)-5-((((2S,3S,5S)-3-acetamido-5-hydroxy-6-(hydroxymethyl)-2,4,6-trimethyl-4-((((2S,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyltetrahydro-2H-pyran-2-yl)methoxy)methyl)tetrahydro-2H-pyran-2-yl)methoxy)methyl)-3,4-dihydroxy-2,4,5,6,6-pentamethyltetrahydro-2H-pyran-2-yl    5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate-   50.    (2S,5S)-3-acetamido-4-((((2R,5S)-5-((((2R,5S)-4,5-dihydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyl-3-((((2S,5S)-3,4,5-trihydroxy-2,3,4,5,6,6-hexamethyltetrahydro-2H-pyran-2-yl)methoxy)methyl)tetrahydro-2H-pyran-2-yl)methoxy)methyl)-3,4-dihydroxy-2,3,4,5,6,6-hexamethyltetrahydro-2H-pyran-2-yl)methoxy)methyl)-5-hydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyltetrahydro-2H-pyran-2-yl    5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate    Structures of iminobiotin compounds are not shown in Table 2.    However, the iminobiotin structures are analogs of the biotin    structure where the biotin group is replaced by an iminobiotin    group. An example is shown below.

In an embodiment of the invention, metal derived targeting agents may bepolymeric or monomeric. Polymeric metal derive targeting agents arefully described in U.S. Pat. No. 7,169,410. Monomeric metal derivedtargeting agents are described in U.S. Pat. No. 4,603,044. Whetherpolymeric or monomeric, the compounds generally comprise a metal(typically purchased as an inorganic salt) that may be selected from thetransition and inner transition metals or neighbors of the transitionmetals. The transition and inner transition metals from which the metalis selected include: Sc (scandium), Y (yttrium), La (lanthanum), Ac(actinium), the actinide series; Ti (titanium), Zr (zirconium), Hf(hafnium), V (vanadium), Nb (niobium), Ta (tantalum), Cr (chromium), Mo(molybdenum), W (tungsten), Mn (manganese), Tc (technetium), Re(rhenium), Fe (iron), Co (cobalt), Ni (nickel), Ru (ruthenium), Rh(rhodium), Pd (palladium), Os (osmium), Ir (iridium), and Pt (platinum).The neighbors of the transition metals from which the metal may beselected are: Cu (copper), Ag (silver), Au (gold), Zn (zinc), Cd(cadmium), Hg (mercury), Al (aluminum), Ga (gallium), In (indium), Tl(thallium), Ge (germanium), Sn (tin), Pb (lead), Sb (antimony) and Bi(bismuth), and Po (polonium). Preferably, the metal is chromium.

Non-limiting examples of useful salts include chromium chloride (III)hexahydrate; chromium (III) fluoride tetrahydrate; chromium (III)bromide hexahydrate; zirconium (IV) citrate ammonium complex; zirconium(IV) chloride; zirconium (IV) fluoride hydrate; zirconium (IV) iodide;molybdenum (III) bromide; molybdenum (III) chloride; molybdenum (IV)sulfide; iron (III) hydrate; iron (III) phosphate tetrahydrate, iron(III) sulfate pentahydrate, and the like.

In addition to a metal, the metal derived targeting agent comprises oneor more complexing agents. A complexing agent is a compound capable offorming a water insoluble coordination complex with the preferred metal.There are several families of suitable complexing agents.

A complexing agent may be selected from the family of iminodiaceticacids of formula (1) wherein R₁ is loweralkyl, aryl, arylloweralkyl, ora heterocyclic substituent.

Suitable compounds of formula (1) include:

-   N-(2,6-diisopropylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(2,6-diethylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(2,6-dimethylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(4-isopropylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(4-butylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(2,3-dimethylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(2,4-dimethylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(2,5-dimethylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(3,4-dimethylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(3,5-dimethylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(3-butylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(2-butylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(4-tertiary butylphenylcarbamoylmethyl)iminodiacetic acid;-   N-(3-butoxyphenylcarbamoylmethyl)iminodiacetic acid;-   N-(2-hexyloxyphenylcarbamoylmethyl)iminodiacetic acid;-   N-(4-hexyloxyphenylcarbamoylmethyl)iminodiacetic acid;-   Aminopyrrol iminodiacetic acid;-   N-(3-bromo-2,4,6-trimethylphenylcarbamoylmethyl)iminodiacetic acid;-   Benzimidazole methyl iminodiacetic acid;-   N-(3-cyano-4,5-dimethyl-2-pyrrylcarbamoylmethyl)iminodiacetic acid;-   N-(3-cyano-4-methyl-5-benzyl-2-pyrrylcarbamoylmethyl) iminodiacetic    acid; and-   N-(3-cyano-4-methyl-2-pyrrylcarbamoylmethyl)iminodiacetic acid and    other derivatives of    N-(3-cyano-4-methyl-2-pyrrylcarbamoylmethyl)iminodiacetic acid of    formula (2),

-   -   wherein R₂ and R₃ are the following:

R₂ R₃ H iso-C₄H₉ H CH₂CH₂SCH₃ H CH₂C₆H₄-p-OH CH₃ CH₃ CH₃ iso-C₄H₉ CH₃CH₂CH₂SCH₃ CH₃ C₆H₅ CH₃ CH₂C₆H₅ CH₃ CH₂C₆H₄-p-OCH₃

Alternatively, the complexing agent may be selected from the family ofimino diacid derivatives of formula (3), wherein R₄, R₅, and R₆ areindependently selected at each occurrence and may be hydrogen,loweralkyl, aryl, arylloweralkyl, alkoxyloweralkyl, and heterocyclic.

Suitable compounds of formula (3) include:N′-(2-acetylnaphthyl)iminodiacetic acid (NAIDA);N′-(2-naphthylmethyl)iminodiacetic acid (NMIDA);iminodicarboxymethyl-2-naphthylketone phthalein complexone; 3(3:7a:12a:trihydroxy-24-norcholanyl-23-iminodiacetic acid; benzimidazolemethyl iminodiacetic acid; and N-(5,pregnene-3-p-ol-2-oylcarbamoylmethyl)iminodiacetic acid.

The complexing agent may also be selected from the family of amino acidsof formula (4),

where R₇ is an amino acid side chain; wherein R₈ may be loweralkyl,aryl, and arylloweralkyl; and wherein R₉ is pyridoxylidene.

Suitable amino acids of the formula (4) are aliphatic amino acids,including, but not limited to: glycine, alanine, valine, leucine,isoleucine; hydroxyamino acids, including serine, and threonine;dicarboxylic amino acids and their amides, including aspartic acid,asparagine, glutamic acid, glutamine; amino acids having basicfunctions, including lysine, hydroxylysine, histidine, arginine;aromatic amino acids, including phenylalanine, tyrosine, tryptophan,thyroxine; and sulfur-containing amino acids, including cystine andmethionine.

The complexing agent may also be selected from amino acid derivativesincluding, but not limited to (3-alanine-γ-amino) butyric acid,O-diazoacetylserine (azaserine), homoserine, ornithine, citrulline,penicillamine and members of the pyridoxylidene class of compounds.Pyridoxylidene compounds include, but are not limited to: pyridoxylideneglutamate; pyridoxylidene isoleucine; pyridoxylidene phenylalanine;pyridoxylidene tryptophan; pyridoxylidene-5-methyl tryptophan;pyridoxylidene-5-hydroxytryptamine; andpyridoxylidene-5-butyltryptamine.

The complexing agent may likewise be selected from the family ofdiamines of formula (6):

wherein R₁₀ is hydrogen, loweralkyl, or aryl; R₁₁ is loweralkylene orarylloweralky; R₁₂ and R₁₃ are independently selected at each occurrenceand may be hydrogen, loweralkyl, alkyl, aryl, arylloweralkyl,acylheterocyclic, toluene, sulfonyl or tosylate.

Examples of suitable diamines of formula (6) include, but are notlimited to, ethylenediamine-N,N diacetic acid;ethylenediamine-N,N-bis(−2-hydroxy-5-bromophenyl) acetate;N′-acetylethylenediamine-N,N diacetic acid; N′-benzoylethylenediamine-N,N diacetic acid;N′-(p-toluenesulfonyl)ethylenediamine-N,N diacetic acid;N-(p-t-butylbenzoyl)ethylenediamine-N,N diacetic acid;N′-(benzenesulfonyl)ethylenediamine-N,N diacetic acid;N′-(p-chlorobenzenesulfonyl)ethylenediamine-N,N diacetic acid;N′-(p-ethylbenzenesulfonyl ethylenediamine-N,N diacetic acid; N′-acyland N′-sulfonyl ethylenediamine-N,N diacetic acid;N′-(p-n-propylbenzenesulfonyl)ethylenediamine-N,N diacetic acid;N′-(naphthalene-2-sulfonyl)ethylenediamine-N,N diacetic acid; andN′-(2,5-dimethylbenzenesulfonyl)ethylenediamine-N,N diacetic acid.

Other, non-limiting examples of complexing compounds or agents includepenicillamine; p-mercaptoisobutyric acid; dihydrothioctic acid;6-mercaptopurine; kethoxal-bis(thiosemicarbazone); Hepatobiliary AmineComplexes, 1-hydrazinophthalazine (hydralazine); sulfonyl urea;Hepatobiliary Amino Acid Schiff Base Complexes; pyridoxylideneglutamate; pyridoxylidene isoleucine; pyridoxylidene phenylalanine;pyridoxylidene tryptophan; pyridoxylidene 5-methyl tryptophan;pyridoxylidene-5-hydroxytryptamine; pyridoxylidene-5-butyltryptamine;tetracycline; 7-carboxy-p-hydroxyquinoline; phenolphthalein; eosin Ibluish; eosin I yellowish; verograffin; 3-hydroxyl-4-formyl-pyrideneglutamic acid; Azo substituted iminodiacetic acid; hepatobiliary dyecomplexes, such as rose bengal; congo red; bromosulfophthalein;bromophenol blue; toluidine blue; and indocyanine green; hepatobiliarycontrast agents, such as iodipamide; and ioglycamic acid; bile salts,such as bilirubin; cholgycyliodohistamine; and thyroxine; hepatobiliarythio complexes, such as penicillamine; p-mercaptoisobutyric acid;dihydrothiocytic acid; 6-mercaptopurine; and kethoxal-bis(thiosemicarbazone); hepatobiliary amine complexes, such as1-hydrazinophthalazine (hydralazine); and sulfonyl urea; hepatobiliaryamino acid Schiff Base complexes, includingpyridoxylidene-5-hydroxytryptamine; andpyridoxylidene-5-butyltryptamine; hepatobiliary protein complexes, suchas protamine; ferritin; and asialo-orosomucoid; and asialo complexes,such as lactosaminated albumin; immunoglobulins, G, IgG; and hemoglobin.

Compositions not Including a Therapeutic Agent

Compositions of the invention may further include one or more associatedtherapeutic agents, however a therapeutic agent need not be present. Inembodiments of the invention that do not have a therapeutic agent, i.e.,compositions that are substantially free of a therapeutic agent, thecomposition comprises constituents prepared from the lipids previouslydisclosed herein, a biotin-derived targeting agent, and gelatin. Theconstituents can comprise a dynamically sized liposome, liposomefragment, and lipid particle, wherein the lipid particle comprises atleast one lipid component and the liposome or liposome fragment cancomprise at least two lipid components.

It has been surprisingly found that administration of the abovedescribed compound has the ability to affect efficient delivery of abiotin and/or a biotin-derived compound, overcoming the solubilityissues plaguing the oral delivery of free biotin and biotin derivedcompounds. Moreover, the composition of the invention has surprisinglyshown that it can affect weight loss in a patient suffering from Type 2diabetes at an effective dosage of biotin at least 5 to 10 times lowerthan previously reported. It has further been surprisingly discoveredthat administration of the above described compound results in drop infasting blood glucose in a patient and reduces HbA1c in Type 2 diabetics(HbA1c, also known as glycated hemoglobin, is a form of hemoglobin usedprimarily to identify the average plasma glucose concentration in apatient over a prolonged period of time).

The above described compound can be administered at any time during theday or night. It can be administered as a single dose, or as multipledoses per 24 hour period, such as 2, 3, 4, 5, 6, 7, 8, 9 or more dosesdepending upon the needs of the patient. In certain embodiments, thecomposition can be administered in 1, 2, 3, or 4 doses.

Dosing can be accomplished before, during, or after a meal.Alternatively, dosing can be accomplished before bed time or shortlyafter waking. In one embodiment, the composition described above can beadministered before breakfast, before lunch, before dinner, and shortlybefore bedtime.

The above described composition can contain an effective amount ofbiotin of from about 1 μg to about 10,000 μg, including all whole andpartial increments there between. In other embodiments, a composition ofthe invention can contain from about 10 μg to about 5,000 μg of aneffective amount of biotin, including all whole and partial incrementsthere between, as well as all ranges included therein. The effectiveamount of biotin in this embodiment of the invention is based on thetotal quantity of biotin-derived targeting agent in the composition. Forexample, a composition comprising 10 mg biotin DHPE would contain aneffective amount of biotin of about 2.6 mg. A composition comprising 5mg of D-biotin as the biotin-derived targeting agent would contain aneffective dose of 5 mg of biotin.

In one embodiment, the composition can contain an effective amount ofabout 78 μg biotin. In other embodiments, the composition can contain aneffective amount of about 78 μg biotin divided by the total number ofdoses given per day. Thus, and by way of example only, a compositiongiven four times a day could contain an effective amount of 19.5 μgbiotin in each dose. (19.5 μg=78 μg biotin/4 dose day).

In another embodiment, the composition can contain an effective amountof about 100 μg biotin. In other embodiments, the composition cancontain an effective amount of about 100 μg biotin divided by the totalnumber of doses given per day. Thus, and by way of example only, acomposition given four times a day could contain an effective amount of25 μg biotin in each dose. (25 μg=100 μg biotin/4 dose day).

In other embodiments, the composition can contain an effective amount ofabout 234 μg biotin, or an effective amount of about 234 μg biotindivided by the total number of doses given per day. In otherembodiments, the therapeutic agent free composition can contain aneffective amount of about 5 mg biotin. In still other embodiments, theabove described composition can contain an effective amount of about 5mg biotin divided by the total number of doses given per day.

In a preferred embodiment, a composition can be formed from lipidcomponents and a biotin-derived targeting agent mixed in accordance withthe following: approximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, approximately 22 mole percentdihexadecyl phosphate, approximately 16 mole percent cholesterol, andabout 1 mole percent of a biotin-derived targeting agent. In certainembodiments the biotin-derived targeting agent is biotin DHPE,biotin-X-DHPE, or D-biotin. The composition can then be mixed with anamount of gelatin.

In another preferred embodiment, a composition can be formed from lipidcomponents and a biotin-derived targeting agent mixed in accordance withthe following: about 56 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, about 21 mole percentdihexadecyl phosphate, about 15 mole percent cholesterol, and about 8mole percent of a biotin-derived targeting agent. In certain embodimentsthe biotin-derived targeting agent is biotin DHPE, biotin-X-DHPE, orD-biotin. An embodiment wherein the biotin-derived targeting agent isbiotin DHPE is shown in the table below.

1,2 distearoyl-sn- glycero-3- dihexadecyl phosphocholine phosphateCholesterol Biotin DHPE MW 790 547 387 940 g 6.04 1.54 0.799 1.047 mg/mL36.3 9.24 4.8 6.3 wt % 64.09 16.31 8.47 11.12 mol % 56.06 20.64 15.148.17

Any of the above described compositions can then be mixed with an amountof gelatin, including from about 200 to about 1,000 mg as well as anywhole or partial increments there between. In particular embodiments,the amount of gelatin is about 230 mg.

In other embodiments, a liquid suspension of the composition can be usedwithout further modification and without being added to gelatin.

While it is preferred that the biotin-derived targeting agent isdelivered as a component of the composition of the invention, in certainembodiments, the biotin-derived targeting agent can be administered to apatient in need thereof free of any additional lipid components. Incertain embodiments, the biotin-derived targeting agent that isadministered is biotin DHPE, or a salt thereof. In other embodiments,the biotin targeting agent is biotin-X-DHPE, or a salt thereof.

The above described composition of the invention (or free biotin-derivedtargeting agent) can also be co-administered with one or more additionaltherapeutic agents useful for inducing weight loss. Examples oftherapeutic agents useful for inducing weight loss include, but are notlimited to, orlistat, sibutramine, phendimetrazine tartrate,methamphetamine, IONAMIN™, phentermine, fenfluramine, dexfenfluramine,chitosan, chromium picolinate, conjugated linoleic acid, green teaextract, guar gum, hoodia, a combination of topiramate and phentermine,a combination of bupropion and zonisamide, a combination of bupropionand naltrexone, a combination of phentermine and fluoxetine, acombination of phentermine and sertraline, a combination of phentermineand citalopram, a combination of phentermine and escitalopram, and acombination of phentermine and trazodone.

It is also contemplated that any of the particular embodiments describedabove can be administered to affect weight maintenance. Moreover, any ofthe embodiments described above can be provided as a food additive,dietary supplement, or as beverage additive. The composition can beadded in any amount to food, taken in any amount as a dietarysupplement, or added to a beverage as appropriate. In certainembodiments, the amount of composition added to a food/beverage or takenas a dietary supplement will not exceed an effective amount of biotin ofabout 10,000 μg/day. In other embodiments, the amount of compositionadded to a food or beverage or taken as a dietary supplement willcontain an effective amount of between about 10 and about 10,000 μgbiotin/day, including all whole and partial increments there between, aswell as all possible sub-ranges.

Compositions of the Invention Including One or More Therapeutic Agents

The constituents of the composition of the present invention mayassociate with one or more therapeutic agents and/or diagnostic agents.Without wishing to be bound by any particular theory, it is believedthat constituents having diameters of 20 nanometers or less aresufficiently small to pass through intercellular gaps, thus enablingtransport of the associated therapeutic agent or diagnostic agent fromthe lumen of the gut into the portal blood.

The associated therapeutic agents and/or diagnostic agents may be boundcovalently or noncovalently to one or more constituents of thecomposition of the present invention. In embodiments of the inventionwherein the associated therapeutic or diagnostic agents are boundcovalently, the associated therapeutic agent or diagnostic agent may bebound to a chemical group that can be functionalized. Examples offunctionalizable groups include, but are not limited to, hydroxy, amino,carboxy, and amido groups.

Examples of therapeutic agents that may be covalently bound to aconstituent of a composition of the present invention includepoly-peptides and/or proteins, such as, but not limited to, D-biotin,GLP-1, insulin, calcitonin, interferon, uricase, tissue plasminogenactivator, thymoglobin, various vaccines, heparin, heparin analogs,antithrombin III, filgrastin, pramilitide acetate, exenatide,epifibatide, and antivenins, blood clotting factors including, but notlimited to, Factors VII and VIII, various small molecules, such as, forexample, D or L thyroxine or serotonin, nucleic acids, DNA or RNAsequences, immunoglobulins, such as, but not limited to, IgG and IgM,and a variety of monoclonal antibodies, such as but not limited to,rituximab, trastuzumab, and glycolipids that act as therapeutic agents,and in addition, other larger proteins, such as, for example, humangrowth hormone (“HGH”), erythropoietin, and parathyroid hormone. Inpreferred embodiments, the covalently attached therapeutic is D-biotin.

Examples of diagnostic agents that may be covalently bound to aconstituent of a composition of the present invention include diagnosticcontrast agents such as, but not limited to, gold and a gadolinium.Other diagnostic agents include radioactive materials such asradioactive isotopes of common atoms including, but not limited to, ¹³C,⁶⁸Ge, ¹⁸F, and ¹²⁵I. These contrast and radioactive agents may becovalently attached to a constituent of the composition directly throughcovalent attachment to a lipid component or targeting agent.Alternatively, and where chemically appropriate, the diagnostic agentmay be bound to a ligand such as DADO (2′-deoxyadenosine), which isitself covalently attached to a lipid component or targeting agent.

Alternatively, and where appropriate chemically, a constituent of acomposition of the invention, may associate with the aforementioneddiagnostic or therapeutic agents via non-covalent interactions.Non-covalent interactions enable compatibility of a constituent of thecomposition of the present invention with a wide variety of diagnosticand therapeutic agents.

Examples of therapeutic agents that can associate with a composition ofthe invention non-covalently include, but are not limited to, D-biotin,insulin, interferon, rituximab, trastuzumab, uricase, tissue plasminogenactivator, thymoglobin, various vaccines, heparin, heparin analogs,anithrombin III, filgrastin, pramilitide acetate, exanatide,epifibatide, antivenins, IgG, IgM, blood clotting Factors VII and VIII,HGH, GLP-1, erythropoietin, parathyroid hormone, serotonin, D- orL-thyroxine, calcitonin, monoclonal antibodies, as well as othertherapeutic peptides. In preferred embodiments, the non-covalentlyassociated therapeutic agent is D-biotin. D-biotin acts at the liver toinduce weight loss.

Association of D-biotin can be achieved via addition of a low molaritysolution of D-biotin to an aqueous suspension of constituents. In thisembodiment, the number of lipid molecules involved in the assembly ofthe constituents far surpasses the number of molecules of D-biotininterlaced and/or combined either on or within the constituents'matricies. This high ratio of constituents to D-biotin is believed tominimize the molecular interactions between D-biotin and theconstituents, insuring that the self-assembly and self-organizationprocess of the constituents of the composition of the present inventionare not disrupted. This high ratio is believed to facilitate theformation of a stable constituent/D-biotin association.

Without wishing to be bound by a particular theory, it is believed thatthe quantity of therapeutic agent(s) associated with a constituent of acomposition of the present invention appears to be a function of loadingtime and lipid concentration. As the lipid component concentration inaqueous media is increased, additional therapeutic agents associate witha constituent of a composition of the present invention. The timerequired for loading the therapeutic agent may be anywhere from severalhours to about one week.

Dosing of a composition comprising D-biotin as the associatedtherapeutic agent can be accomplished before, during, or after a meal.Alternatively, dosing can be accomplished before bed time or shortlyafter waking. In one embodiment, the composition described above can beadministered before breakfast, before lunch, before dinner, and shortlybefore bedtime.

The amount of associated D-biotin can be from about 1 to about 10,000 μgof biotin, including all whole and partial increments there between. Inother embodiments, the amount of associated D-biotin can be about 10 μgto about 5,000 μg, including all whole and partial increments therebetween, as well as all ranges included therein.

In one embodiment, the composition can contain about 78 μg D-biotin. Inother embodiments, the composition can contain 78 μg D-biotin divided bythe total number of doses given per day. Thus, and by way of exampleonly, a composition given four times a day could contain 19.5 μgD-biotin in each dose. (19.5 μg=78 μg D-biotin/4 dose day).

In other embodiments, the composition can contain about 234 μg D-biotin,or about 234 μg D-biotin divided by the total number of doses given perday. In other embodiments, the therapeutic agent free composition cancontain 5 mg D-biotin. In still other embodiments, the therapeutic agentfree composition can contain 5 mg D-biotin divided by the total numberof doses given per day.

In a preferred embodiment, a composition with D-biotin as the associatedtherapeutic agent can be formed from lipid components and, optionally, abiotin-derived targeting agent mixed in accordance with the following:approximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, approximately 22 mole percentdihexadecyl phosphate, approximately 16 mole percent cholesterol, andabout 1 mole percent of a biotin-derived targeting agent (if present).In certain embodiments the biotin-derived targeting agent is biotinDHPE, biotin-X-DHPE, or D-biotin.

A composition having D-biotin as the associated therapeutic agent canalso be co-administered with one or more additional therapeutic agentsuseful for inducing weight loss. Examples of therapeutic agents usefulfor inducing weight loss include, but are not limited to, orlistat,sibutramine, phendimetrazine tartrate, methamphetamine, IONAMIN™,phentermine, fenfluramine, dexfenfluramine, chitosan, chromiumpicolinate, conjugated linoleic acid, green tea extract, guar gum,hoodia, a combination of topiramate and phentermine, a combination ofbupropion and zonisamide, a combination of bupropion and naltrexone, acombination of phentermine and fluoxetine, a combination of phentermineand sertraline, a combination of phentermine and citalopram, acombination of phentermine and escitalopram, and a combination ofphentermine and trazodone.

It is also contemplated that a composition of the invention havingD-biotin as the associated therapeutic agent can be administered toaffect weight maintenance, or provided as a food additive, dietarysupplement, or as beverage additive. The composition can be added in anyamount to food, taken in any amount as a dietary supplement, or added toa beverage as appropriate. In certain embodiments, the amount ofcomposition added to a food/beverage or taken as a dietary supplementwill not exceed an effective dose of D-biotin of about 10,000 μg/day. Inother embodiments, the amount of composition added to a food or beverageor taken as a dietary supplement will include between about 10 and about10,000 μg D-biotin/day, including all whole and partial increments therebetween, as well as all possible sub-ranges.

The low concentration of therapeutic agent relative to the concentrationof the constituents of the composition of the present invention isunique among lipid particle delivery systems. Typically, liposome orliposome-like delivery systems have employed a much larger quantity oftherapeutic agent. The efficacy this embodiment of the presentcombination shows that it is possible to utilize less therapeutic agentwhile still obtaining a pharmacologically desirable result in thepatient. This embodiment of the invention therefore provides anadvantageous therapeutic option.

In other embodiments the addition of a higher concentration oftherapeutic agent may be both desirable and advantageous. Theconstituent members of a composition of the present invention arecapable of associating with, and tolerating, higher molarity solutionsof any given therapeutic agent.

Insulin can also be therapeutic agent associated with a composition ofthe invention. FIG. 1 illustrates a constituent/HTM/insulin construct.Insulin molecules bind to the surface of the constituent vianon-covalent electrostatic interactions.

Serotonin, like insulin and D-biotin, may also be delivered to the liverutilizing a constituent/HTM complex according to the invention.Serotonin acts jointly with insulin at the level of the liver toactivate hepatic glucose storage during a portal (oral) glucose load. Inorder to achieve the desired effect, serotonin must be delivered to theliver. Non-targeted serotonin, introduced via injection or oral deliveryin pharmacologically acceptable doses cannot effectively induce thedesired activity. Therefore, an embodiment of the invention comprising aconstituent/HTM/serotonin construct provides a highly desirable deliverymechanism for this important gluco-regulatory hormone. In an embodimentof the invention designed for the delivery of serotonin, the lipidcomponents selected to form the constituents of the composition includeapproximately 62 mole percent,1,2-distearoyl-sn-glycero-3-phosphocholine, approximately 22 molepercent dihexadecyl phosphate, approximately 16 mole percent cholesteroland about 1 mole percent of a targeting agent.

Calcitonin is a hormone that regulates bone metabolism. Due to the highprevalence of diseases such as osteoporosis, an oral formulation of thishormone is highly desirable. Presently calcitonin is only deliverablevia injection. In an embodiment of the invention designed for thedelivery of calcitonin, the lipid components selected to form theconstituents of the composition including calcitonin includeapproximately 62 mole percent,1,2-distearoyl-sn-glycero-3-phosphocholine, approximately 22 molepercent dihexadecyl phosphate, and approximately 16 mole percentcholesterol.

GLP-1 is a peptide that acts at both the liver and pancreas. In theliver, GLP-1 acts to stimulate glycogen accumulation during a meal.However, prior art administration methods where GLP-1 is administeredorally evidence poor bioavailability and reduced efficacy upon oraldosing. In an embodiment of the present invention, GLP-1 associates witha constituent of a composition of the invention form aconstitutent/GLP-1 construct. The constituent/GLP-1 construct mayfurther include a targeting agent. Preferably, the lipid componentsselected to form the constituents of the composition including GLP-1include approximately 62 mole percent1,2-distearoyl-sn-glycero-3-phosphocholine, approximately 22 molepercent dihexadecyl phosphate, and approximately 16 mole percentcholesterol.

Thyroxine, like insulin, is also not generally orally bioavailable. Inan embodiment of the invention, though, thyroxine may associate with aconstituent of a composition of the invention forming aconstituent/thyroxine construct. Preferably, the lipid componentsselected to form the constituents of the composition including thyroxineinclude approximately 62 mole percent,1,2-distearoyl-sn-glycero-3-phosphocholine, approximately 22 molepercent dihexadecyl phosphate, approximately 16 mole percentcholesterol, and approximately 1 mole percent Biotin DHPE.

Although the invention has been described in terms of specifictherapeutic agent/constituent constructs, any of the therapeutic agentsdescribed herein may associate with a constituent of the invention toform a therapeutic agent/constituent construct.

Covalently Attached Therapeutic Agents

In certain embodiments of the invention, the therapeutic agent may becovalently attached to a lipid component of the invention. Typically,however, the covalent attachment of the therapeutic agent to the lipidcomponent is not direct, but is mediated by a linker of the form—C(O)(CH₂)_(n)SR, wherein an amide, ester, or thioamide bond is formedbetween the therapeutic agent and the linker. Preferably, n is aninteger between 1 and 10. Even more preferably, n is 1, 2, or 3. Whenthe linker is being attached to the therapeutic agent, R is typically aprotecting group such as —C(O)CH₃. Other appropriate thiol protectinggroups may be found in Green's Protective Groups in Organic Synthesis,Wuts, et al, 4^(th) edition, 2007.

After the linker is bound to the therapeutic agent, the protectinggroup, R, is removed from the linker to reveal a free thiol group.Preferably, the protecting group is removed under conditions that do notperturb the now attached therapeutic agent. This thiol may then undergoa Michael reaction with a lipid component such as MPB-PE to form a thioether. Preferably, lipid component MPB-PE is already incorporated into aconstituent of a compound of the invention, however, the linker may bebound to the MPB-PE prior to its incorporation a constituent of theinvention. The order of reactions will depend upon the therapeuticagent's ability to tolerate certain reaction conditions. In the case ofcomplex proteins which may denature at high temperatures, it ispreferable to perform the Michael reaction after MPB-PE has beenincorporated into a constituent of the compound of the invention.

In an example of a covalent interaction, IgG was covalently linked to alipid component of a constituent of the invention to form aconstituent/IgG construct. IgG is an antibody that is not normallyorally bioavailable. In this embodiment of the invention, the lipidcomponents selected to form the constituents of the constituent/IgGconstruct include approximately 68 mole percent1,2-dipalmitoyl-sn-glycero-3-phosphocholine, approximately 18 molepercent dihexadecyl phosphate, approximately 9 mole percent cholesterol,and approximately 3 mol percent MPB-PE.

In order to form the constituents of the invention,1,2-dipalmitoyl-sn-glycero-3-phosphocholine, dihexadecyl phosphate, andcholesterol were microfluidized as set forth earlier herein to formconstituents with an upper size limit of between 50 and 60 nanometers.This suspension of constituents was then transferred to a round bottomflask that had been coated with a thin film of MPB-PE. The suspensionwas heated to about 62° C., with the temperature not falling below 60°C. or exceeding 65° C. The heated suspension was subsequently stirredfor 15 minutes until all of the MPB-PE had been incorporated into theconstituents of the invention.

Separately, IgG was reacted with a 10 fold excess of linker precursor I(R═CH₃C(O), n=1), below, to form II. Compound II was then purified usinga 2.5×25 cm Sephadex G-25 column equilibrated with 18 mM phosphatebuffer plus 1.0 mM EDTA buffer at pH 7.4.

Next, the acetyl protecting group on compound II was removed by stirringcompound II with 50 mM hydroxylamine hydrochloride in 18 mM sodiumphosphate buffer containing 1.0 mM EDTA (pH 7.4) for 2 hours at ambienttemperature. The resulting free thiol, III, was purified on 2.5×25 cmSephadex G-25 column, as set forth for compound II.

Immediately following purification, 200μ-moles of compound III was mixedwith 10 ml of the constituent solution prepared earlier. The reactionmixture was stirred for 15 minutes, during which time compound IIIunderwent a Michael reaction with the maleimide functionality of theMBP-PE incorporated into the constituents of the invention. Theconjugation reaction was stopped, and excess III removed, by theaddition of a 50× molar excess of N-ethylmaleimide.

Although the above example was described with respect to IgG, it isequally applicable to any therapeutic agent with a basic nitrogen orfree hydroxyl group, or other functionalizable group, able to bind tothe linker or linker precursor.

Stability

Although constituent members of a composition of the present inventionare formulated in aqueous media, the constituent members of thecomposition do not exhibit long term stability in water. Specifically,water aids hydrolysis of any acyl chains present in any of the lipidcomponents of the compositional constituents. The aqueous environmentalso allows for the ready oxidation of any unsaturated acyl chainspresent in any of the lipid components. In a preferred embodiment of thepresent invention, the constituents of the composition of the presentinvention may be protected for long term storage via interaction with aproteoglycan such as a modified collagen, known generically as drygranulated gelatin. Dry granulated gelatin, when contacted with anaqueous suspension of constituents, reacts with water, stabilizes theconstituents, and forms a composition of the present invention.

The reaction of dried granulated gelatin with an aqueous suspension ofconstituents of a composition of the present invention results in asemi-solid colloidal gel that shields the constituents from directinteraction with water. Any water not associated with gelatin is slowlyevaporated via refrigerated storage at about 2° to about 8° C. The watermay, however, be removed via techniques including, but not limited,freeze drying and spray drying.

This results in a pellet like “dry” constituent/gelatin complex which isthe composition of the invention. In the composition, the constituentelements are partially dehydrated in a reversible manner and sequesteredby the proteinaceous lattice of dry gelatin. This sequestration isenabled by structured water, structured lipid and structured gelatin allinteracting through hydrogen bonding, ionic bonding, van der Waal'sinteractions, and hydrophobic bonding between the lipid components,water, and protein structures, i.e., insulin. This evidences thatgelatin is not acting as an emulsifying or suspending agent. As aresult, the “dry” pellet is stable for long term storage because theactivity of water has been mitigated. These pellets can be furtherprocessed to a granulated or free-flowing powder for final capsulefilling or tabletting, while maintaining their stability.

Upon oral administration to a patient, the “dry” pellet becomes hydratedand once again assumes a semi-solid colloidal gel state. Upon furtherexposure to the gastric environment, the gel becomes liquid as gelatinis solubilized. Once the gelatin is completely solubilized, theconstituent members of the composition of the invention rehydrate,resulting in the formation of a new suspension of constituents withinthe gastric environment. The reconstituted constituents may then beabsorbed into the portal blood flow.

It is important to realize that the role of gelatin in this aspect ofthe invention is as an active stabilizer of the composition and not aninert filler as is commonly found in oral formulations of many otherpharmaceutical compositions. That said, the additional use of gelatin asan inert filler in addition to the aforementioned use is alsocontemplated.

Although gelatin is used in a preferred embodiment of the invention,other gelatin like compounds may be used as well. Examples of agentsthat will act as active stabilizers include, but are not limited to,acacia (gum arabic), agar (agar-agar; vegetable gelatin; gelosa; Chineseor Japanese gelatin), alginic acid, sodium alginate (alginic acid;sodium salt; algin; Manucol; Norgine; Kelgin), carbomer(carboxypolymethylene), carrageenan, carboxymethylcellulose sodium(carbose D; carboxymethocel S; CMC; cellulose gum), powdered cellulose(Degussa), hydroxyethyl cellulose (cellulose; 2-hydroxyethyl ether;Cellosize; Natrosol), hydroxypropyl cellulose (cellulose;2-hydroxypropyl ether; Klucel), hydroxypropyl methylcellulose(cellulose; 2-hydroxypropyl methyl ether), methycellulose (cellulose;methyl ether Methocel), povidone (2-pyrrolidinone; 1-ethenyl-;homopolymer; polyvinylpyrrolidone), tragacanth (gum tragacanth; Hog Gum;Goat's Thorn), and xanthan gum (Keltrol). Like gelatin, and whereappropriate, these compounds may also be used as inert fillers.

Formulations

A formulation of a composition of the invention—hereinafter“composition”—for oral administration may be prepared, packaged, or soldin the form of a discrete solid dose unit including, but not limited to,a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, eachcontaining a predetermined amount of the active ingredient. Otherformulations suitable for oral administration include, but are notlimited to, a powdered or granular formulation, aqueous suspensions, oremulsions.

A tablet comprising the composition of the present invention, forexample, be made by compressing or molding the composition optionallywith one or more additional ingredients. Compressed tablets may beprepared by compressing, in a suitable device, the composition in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, the composition, a pharmaceuticallyacceptable carrier, and at least sufficient liquid to moisten themixture.

Pharmaceutically acceptable excipients used in the manufacture oftablets include, but are not limited to, inert diluents, granulating anddisintegrating agents, binding agents, and lubricating agents. Knowndispersing agents include, but are not limited to, potato starch andsodium starch glycollate. Known surface active agents include, but arenot limited to, sodium lauryl sulphate. Known diluents include, but arenot limited to, calcium carbonate, sodium carbonate, lactose,microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of thecomposition. By way of example, a material such as glyceryl monostearateor glyceryl distearate may be used to coat tablets. Further by way ofexample, tablets may be coated using methods described in U.S. Pat. Nos.4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlledrelease tablets. Tablets may further comprise a sweetening agent, aflavoring agent, a coloring agent, a preservative, or some combinationof these in order to provide pharmaceutically elegant and palatablepreparation.

Hard capsules comprising the composition may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, kaolin or celluloseacetate hydrogen phthalate.

Soft gelatin capsules comprising the composition may be made using aphysiologically degradable composition, such as gelatin.

Liquid formulations of the composition which are suitable for oraladministration may be prepared, packaged, and sold either in liquid formor in the form of a dry product intended for reconstitution with wateror another suitable vehicle prior to use, subject to the stabilitylimitations disclosed earlier.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the constituents in an aqueous vehicle. Aqueous vehiclesinclude, for example, water and isotonic saline. Oily vehicles may onlybe used to the extent that such solvents are not incompatible with theconstituents of the composition of the present invention. To the extentthat an oily suspension is not incompatible with the constituents of thecomposition of the present invention, an oily suspension may furthercomprise a thickening agent.

Liquid suspensions may further comprise one or more additionalingredients to the extent that said ingredients do not disrupt thestructures of the constituents of the composition of the invention.Examples of additional ingredients include, but are not limited to,suspending agents, dispersing or wetting agents, emulsifying agents,demulcents, preservatives, buffers, salts, flavorings, coloring agents,and sweetening agents.

Known suspending agents include, but are not limited to, sorbitol syrup,sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, andcellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose.

Known emulsifying agents include, but are not limited to acacia. Knownpreservatives include, but are not limited to, methyl, ethyl, orn-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Knownsweetening agents include, for example, glycerol, propylene glycol,sorbitol, sucrose, and saccharin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous suspension orsolution by addition of an aqueous vehicle thereto. Each of theseformulations may further comprise one or more of dispersing or wettingagent, a suspending agent, and a preservative. Additional excipients,such as fillers and sweetening, flavoring, or coloring agents, may alsobe included in these formulations.

Methods of Treating Diseases

Diseases, such as diabetes may be treated by orally administering acompound of the invention wherein insulin is the associated therapeuticagent. Similarly, diabetes may be treated by orally administering acompound of the invention wherein insulin is the associated therapeuticand wherein another form of insulin is co-administered. Routes ofco-administration include, but are not limited to, oral administration,intramuscular injection, inhalation, intravenous injection,intra-arterial injection, as well as any other form of administration.

Although a physician will be able to select the appropriate dose for agiven patient, the range of doses that may be delivered in a givenformulation of a compound of the invention is from about 1 to about 40units, but may be 5, 10, 15, 20, 25, 30, or 35 units. A givenformulation may, however, contain any whole or partial integertherebetween and may exceed 40 units.

Kits

The invention also includes a kit comprising a composition of theinvention and an instructional material which describes administeringthe composition to a mammal. In another embodiment, this kit comprises acomposition of the invention, insulin for co-administration, as well asinstructional material which describes the co-administration process.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the composition of the inventionin the kit for effecting alleviation of the various diseases ordisorders recited herein.

Optionally, or alternatively, the instructional material may describeone or more methods of alleviating the diseases or disorders in a cellor a tissue of a mammal. The instructional material of the kit may, forexample, be affixed to a container which contains the invention or beshipped together with a container which contains the invention.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the instructional material and thecompound be used cooperatively by the recipient.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Experiment 1 Administration of Compositions not Containing a TargetingAgent

A composition whose constituent members were created from a mixture oflipid components comprising approximately 62 mole percent1,2-distearoyl-sn-glycero-3-phosphocholine, approximately 22 molepercent dihexadecyl phosphate, approximately 16 mole percentcholesterol, and no targeting agent was prepared according to themicrofluidization procedure generally described herein. A known portionof the lipid component comprised ¹⁴C labeled phospholipid. Followingfiltration through a 0.2 micron filter, the average constituent size wasless than 100 nm as measured with a Coulter Sub-micron Particle SizeAnalyzer.

A 10 mg/kg body weight sample of the composition (containing 85,000 cpmof ¹⁴C radio-label) was then injected into the duodenum of ananesthetized 230 gram fasted, but otherwise normal, rat. Blood was takenfrom the portal and femoral veins at 15 and 30 minutes post-dosing forcounting (FIG. 2). At 30 minutes post-dosing, the rat was sacrificed andrepresentative samples of blood, liver, and spleen were removed foranalysis (FIG. 3).

Labeled constituents, as measured by ¹⁴C, were found in both portal andfemoral blood of the rat. The portal blood levels of ¹⁴C labeledconstituents were higher than the femoral blood levels (FIG. 2). At 30minutes post-dosing, approximately 15% of the constituents that wereinjected into the gut were found in the blood. Approximately 4% of thecounts were found in the liver and about 1% were found in the spleen.Considering the relative sizes of the liver and spleen, the splenicuptake was much higher than liver uptake on a weight basis.

Experiment 2 Hepatocyte Targeting

To demonstrate the absorption of the composition from the gut, acomposition comprising insulin and constituents generated from a mixtureof lipid components comprising approximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, approximately 22 mole percentdihexadecyl phosphate, approximately 16 mole percent cholesterol, andapproximately 1 mole percentpoly[Cr-bis(N-2,6-diisopropylphenylcarbamoylmethyl iminodiacetic acid)](wherein a known portion of the phospholipid component comprised ¹⁴Clabeled phospholipid) was prepared as recited in the general preparationdisclosed herein. Prior to dosing, the labeled composition to rats, therats were fasted from food for 24 hours and from water for 4 hours. Thefasted rats were then permitted to drink water from a graduated waterbottle containing the composition. The drinking water bottle was removedfrom the cage after 15 minutes, the amount of water ingested from thedrinking bottle was measured, and the amount of composition ingested wascalculated. The rats' blood was sampled at 15, 30, and 45 minutes andthe radiolabel in each sample was counted (FIG. 4). At 45 minutes therats were sacrificed and the livers were counted for radio-label (FIG.5).

As is shown in FIG. 4, approximately 8% of the ingested dose was foundin the rats' blood 15 minutes after the water had been removed from thecage. The quantity of constituents in the rats' blood remained constantbetween 15 and 45 minutes. Liver uptake was approximately 8% at 45minutes. Splenic uptake at 45 minutes was approximately 1% of theingested dose (FIG. 5). The total absorption was approximately 17%(including blood, liver, and spleen).

Experiment 3 Hepatocyte Targeting with a Composition inAlloxan-Streptozotocin Treated Mice

Mice used in the present experiment were made diabetic by administeringstreptozotocin and alloxan. The diabetic animals were then divided intotwo groups. The control group (11 mice) was orally dosed with regularinsulin. The experimental group (7 mice) was orally dosed with acomposition comprising insulin and constituents generated from a mixtureof lipid components comprising approximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, approximately 22 mole percentdihexadecyl phosphate, approximately 16 mole percent cholesterol, andapproximately 1 mole percentpoly[Cr-bis(N-2,6-diisopropylphenylcarbamoylmethyl iminodiacetic acid)](wherein a known portion of the phospholipid component comprised ¹⁴Clabeled phospholipid). Dosing was accomplished utilizing the waterbottle dosing method described in Experiment 2.

After being made diabetic, rats in both groups were treated identicallyover a 7 day period and fed with plain food and plain water. Followingthis 7 day period, rats in the control group were treated for anadditional 7 day experimental period with food and regular insulin inthe available drinking water at 0.1 U/ml. Over the same 7 dayexperimental period, the experimental group was fed regular food withthe composition of the invention available in the drinking water at 0.1U/ml. At the end of each 7-day period, blood glucose was measured in atail-vein sample of blood by a Beckman Blood Glucose Analyzer.

The pharmacologic efficacy of orally administered insulin in the groupdosed with the above described composition is shown in FIG. 6. Micereceiving the composition had a statistically significant reduction inblood glucose on day seven (p<0.01) compared to mice receiving regularinsulin, whose blood glucose was not altered at all.

Example 4 In Vivo Administration of Serotonin

The hepatic action of a composition comprising serotonin andconstituents generated from a mixture of lipid components comprisingapproximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, approximately 22 mole percentdihexadecyl phosphate, approximately 16 mole percent cholesterol, and 1mole percent of poly[Cr-bis(N-2,6-diisopropylphenylcarbamoylmethyliminodiacetic acid)] was demonstrated in a type 2 diabetic dog (truncalvagotomy). The dog was fasted, and then anesthetized. Blood samplingcatheters were placed in the hepatic and portal veins to enablesimultaneous blood sampling. Glucose was infused into the portal systemat a rate of 0.5 g/kg/hour. Next, the above described composition wasadministered intraduodenally in a single dose of 30 μg/kg body weight.Results are depicted in FIG. 7 and demonstrate that serotonin (alsoreferred to as 5-hydroxytryptamine or 5-HT), administeredintraduodenally as a composition of the invention is effective at lowdoses in converting a type 2 diabetic dog from hepatic glucose output touptake during a portal glucose load.

Example 5 In Vivo Administration of Calcitonin

Normal, fasted, control rats were given a dose of salmon calcitonin viasubcutaneous injection such that an initial 10% reduction in bloodcalcium was observed. Blood calcium levels were then measured for sixhours post injection. An experimental group of rats was given the sameeffective dose of calcitonin by oral gavage, in the form of acomposition comprising calcitonin and constituents generated from amixture of lipid components comprising approximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, approximately 22 mole percentdihexadecyl phosphate, and approximately 16 mole percent cholesterol.Blood calcium levels were followed for six hours (FIG. 8). A bloodcalcium reduction of up to 20% was observed in the non-control rats.This difference was statistically significant (FIG. 8).

Example 6 Clinical Trial with Targeted Insulin in Type 2 DiabetesMellitus Subjects

Capsules containing a composition of the invention were prepared. Thecomposition comprised insulin as the therapeutic agent, gelatin, andconstituents generated from a mixture of lipid components comprisingapproximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, approximately 22 mole percentdihexadecyl phosphate, approximately 16 mole percent cholesterol, andabout 1 mole percent of the sodium salt of Biotin-HDPE. Each capsulecontained 2 U of insulin.

Six well characterized Type 2 diabetes patients participated in thecontrolled study. The patients were maintained on their customary Type 2oral anti-diabetes therapy. Study participants were also given eitherplacebo capsules or the above described capsules 30 minutes before a 60gram carbohydrate meal at breakfast, lunch and dinner. Blood sampleswere drawn at frequent intervals over a 13 hour period and theIncremental Area Under the Curve for the blood glucose values wascalculated for each subject.

At 0.1 U/kg body weight/meal, the same dose that is frequently used withsubcutaneous injection of insulin at a given meal, a statisticallysignificant reduction in AUC for each of the three meals was observed.FIG. 10 depicts the results of the trial in graphical format.

Example 7 Insulin Concentration

Insulin U-500 contains 500 units of insulin/ml=0.5 units/1 μl

-   -   Add 3.36 ml of U-500 insulin to 70 ml of constituent suspension        in 18 mM phosphate buffer @ pH 7.01.    -   (3,360 μl)*(0.5 units of insulin/μl)=1,680 units of insulin        total in 73.36 ml    -   (1,680 units of insulin)/(73.36 ml)=22.9 units of        insulin/ml—or—34.35 units of insulin/1.5 ml    -   Load insulin for 21 hours;    -   Post loading, chromatograph 1.5 ml of sample over a 1.5 cm×25 cm        column with Sepharose CL-6B gel equilibrated with 18 mM        phosphate buffer @ pH 7.01    -   0% of free insulin recovered from column; The recovery of 0% of        the total loaded insulin implies that 100% of the total “loaded”        insulin is associated with a constituent of the composition.    -   34.35 units of insulin×100%=34.35 units of insulin bound or        associated with the constituents of the invention.        FIG. 11 depicts the above described chromatography. A trace        showing the elution time of free insulin is included for        purposes of comparison. As can be seen from the chromatogram,        insulin is associated with the constituents of the invention and        no free insulin is in solution. A preservative included with        insulin does not associate with the constituents of the        composition of the invention and is visible in the chromatogram.

Example 8 Oral Delivery of GLP-1

Rats were fasted overnight. Subsequently, 800 mg each of alloxan andstreptozotocin were dissolved in 40 mL of PBS (pH 7, 0.01 M). The fastedrats were then treated immediately with a 0.5 mL IP dose to induceinsulin deficiency. The animals were then stabilized overnight withwater and food. Following stabilization, the rats were fasted overnightto deplete liver glycogen.

Subsequently, 1.5 g glucose/kg body weight and GLP-1 in the form of aGLP-1/constituent construct were simultaneously administered via oralgavage. The constituents were prepared from a mixture of lipidcomponents comprising approximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, approximately 22 mole percentdihexadecyl phosphate, and approximately 16 mole percent cholesterol(“associated GLP-1”). In separate experiments, the amount of associatedGLP-1 was varied. Liver glycogen was measured chemically at 2 hours postdosing.

As a control, unassociated GLP-1 was gavaged in place of associatedGLP-1. In a separate control, GLP-1, in a dose similar to that orallygavaged, was injected intraperitoneally. As is shown in Table 3, below,substantially enhanced oral efficacy was observed for the associatedGLP-1 versus non-associated GLP-1.

TABLE 3 Dose GLP-1 Liver Glycogen Treatment mg/rat mg/g liver ControlOral GLP-1 0.01 40 ± 22 Intraperitoneal GLP-1 0.01 59 ± 44 OralAssociated GLP-1 0.005  73 ± 56* Oral Associated GLP-1 0.01  90 ± 75* *p= 0.05 compared to Control Oral GLP-1

Example 9 Oral IgG

Human IgG antibodies were covalently attached to a constituent of theinvention, as described previously herein (“covalent IgG”).Subsequently, eight 250 gram laboratory rats were prepared withintra-duodenal catheters for the administration of covalent IgG. Afteran overnight fast, 5 ug of covalent IgG was infused into the duodenalcatheter. The catheter was subsequently washed with 0.5 ml buffer. Bloodsamples were taken at 15, 30, 60 and 120 minutes to assay the plasmaconcentration of human IgG antibodies by ELISA reaction.

In a control experiment, 5 ug of free IgG was infused into the duodenalcatheter. The catheter was subsequently washed with 0.5 ml buffer. Bloodsamples were taken at 15, 30, 60 and 120 minutes to assay the plasmaconcentration of human IgG antibodies by ELISA reaction. The results ofboth studies are shown in FIG. 12.

As can be seen in FIG. 12, covalent IgG provided enhanced plasmaconcentration of human IgG (AUC) as compared to free IgG. Likewise,covalent IgG enhanced Tmax—the time to maximum concentration, andCmax—the maximum plasma concentration observed upon dosing. The enhancedefficacy of covalent IgG, as compared to free IgG, thus demonstrates theability of a compound of the invention to enhance oral absorption ofvery large proteins into the systemic circulation.

Example 10 Oral Thyroxine

Thyroxine is known to lower blood cholesterol and triglyceride levels.However, at the doses required to treat high cholesterol andtriglyceride, thyroxine causes hyperthyroidism as an unwanted sideeffect. The goal of this study was to demonstrate that orallyadministered targeted thyroxine associated with a compound of theinvention would act at the liver with the result of lowering bloodlipids without inducing the unwanted hyperthyroidism.

Normal laboratory mice, on high caloric diets, were administered loworal doses (0.2 to 1.0 μg) thyroxine in the form of a compositioncomprising thyroxine and constituents generated from a mixture of lipidcomponents comprising approximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, approximately 22 mole percentdihexadecyl phosphate, approximately 16 mole percent cholesterol, andapproximately 1 mole percent of the sodium salt of Biotin-HDPE, aliver-targeting agent.

The mice, in groups of 4, were dosed daily by oral gavage for one weekin a dose response study. Blood cholesterol and triglycerides weremeasured after one week treatment. Baseline values for cholesterol andtriglycerides for all the groups were similar. The dose responses, shownin FIG. 13, demonstrates the efficacy of orally administered, hepatictargeted thyroxine associated with a composition of the invention. Bloodlevels of thyroid hormone did not increase with the dosing of hepatictargeted oral thyroxine, demonstrating the safety of the product.

Other published studies (Erion, M., et al., PNAS Sep. 25, 2007 vol 104,#39, pp 15490-15495) with hepatic targeted thyroxine analogs requireddoses at least 10 fold higher than those described herein to elicitsimilar reductions in blood cholesterol and triglycerides.

Example 11 Oral Interferon

A composition was prepared comprising interferon-α as the therapeuticagent and constituents generated from a mixture of lipid componentscomprising approximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine, approximately 22 mole percentdihexadecyl phosphate, approximately 16 mole percent cholesterol, andabout 1 mole percent of the sodium salt of Biotin-HDPE.

Six patients with Hepatitis C, genotype 3, were treated with an aqueoussuspension of the above described composition and Ribivirin daily for 8weeks. The interferon-α dose in the aqueous suspension of thecomposition was 60,000 Units/day.

Hepatitis C viral loads were measured at the beginning of the study,then at weeks 1, 2, 4, and 8. See FIG. 14. The data demonstrates theability of the aqueous suspension of a composition of the invention tolower viral load with a minimal dose of interferon. Side effects werelikewise minimized.

Example 12 Clinical Trial in Type 2 Diabetes Mellitus Subjects

An 18 week study was designed to review the efficacy of a compound ofthe invention for controlling blood glucose levels in Type 2 diabetics.Data for patients administered a composition comprising insulin, i.e.the non-control group is not reported herein. Data for the controlgroup, i.e. patients administered a composition of the invention withoutinsulin, is presented below.

Capsules containing a composition of the invention were prepared for thecontrol group. The composition provided to the control group comprisedgelatin (230 mg), approximately 61 mole percent 1,2distearoyl-sn-glycero-3-phosphocholine (3.36 mg), approximately 22 molepercent dihexadecyl phosphate (0.86 mg), approximately 16 mole percentcholesterol (0.44 mg), and about 1 mole percent of the sodium salt ofBiotin DHPE (0.075 mg).

A variable Baseline Stabilization Phase of up to 12 weeks was deployedto stabilize patients on background metformin therapy. All patientspreviously treated with metformin therapy for a minimum of 12 weeksdirectly entered into the study at the end of Baseline StabilizationPeriod 7-point glucose testing described below. Naïve treatment diabeticpatients or patients on metformin in combination with other OADs wereplaced on metformin alone and stabilized on the mono-therapy for 12weeks. At the end of the Baseline Stabilization Period, the patientunderwent two consecutive days of 7-point blood glucose tests (Days −1and 0). The 7-point glucose tests results were recorded on PatientDiaries.

Patients entered the “Treatment Phase” of the study once they optimizedtheir metformin dosing regimen. This was Day 1 of the Treatment Phase.At the Day 1 Visit, Baseline HbA1c levels were obtained. Following theBaseline Stabilization Phase, patients were administered the abovedescribed capsules. Patients on the “low dose” (78 μg biotin daily) weregiven one capsule per dose, with four doses daily (10-30 minutes beforebreakfast, 10-30 minutes before lunch, 10-30 minutes before dinner, and10-30 minutes before bed time). Patients on the “high dose” (234 μgbiotin daily) were given three capsules per dose, with the same dosingschedule as the low dose patients. During the Treatment Phase, patientswere instructed to follow the ADA “weight maintenance diet”.

At the end of Week 2 (Day 14), patients performed a 7-point glucosetest. At the end of Week 4 (Days 27 and 28), patients performed 7-pointglucose tests and returned to the clinic for the Week 4 visit (Day 29).At this visit, an HbA1c level was obtained. Patients continued in thestudy with site visits at Week 8 (Day 57), with 7-point glucose testperformed on Days 55 and 56 and Week 12 (Day 85), with 7-point glucosetests on Days 83 and 84. On Days 57 and 85, an HbA1c level was obtained.The Week 18 Visit was conducted on Day 127 with the 7-point glucose testperformed on Days 125 and 126. An HbA1c level was also obtained at thisvisit. After 18 weeks, patients dosed with a therapeutic agent freecomposition of the invention experienced a reduction in fasting bloodglucose of about 7 mg/dl (low dose) and about 35 mg/dl (high dose). See,for example, FIG. 15. All patients in the study, at both the high andlow dose, showed reduced HbA1c concentrations at 18 weeks, as shown inFIG. 16.

Data obtained at the 18 week time point (intent to treat population) forthe 7-point glucose test showed a slight increase for the low dosepopulation, but a statistically significant reduction of about 10 mg/dlin the high dose population. The data is represented graphically in FIG.19.

In addition to the above, after 18 weeks, patients on the low dose, onaverage, lost about 1.2% of their total body mass, while patients on thehigh dose lost approximately 1.9% of their total body mass. About 69% ofsubjects in the study observed some weight loss. These data are showngraphically in FIG. 17.

When the 18 week weight loss data was analyzed to exclude those patientswho lost no weight, the average weight loss for low dose patients wasapproximately 2.5% of total body mass, while high dose patients lost, onaverage, about 3.8% of their total body mass. These data are representedgraphically in FIG. 18.

Various curves extrapolating total weight loss over 1 year were createdbased on the 18 week weight loss data. See FIG. 20. It is evident, basedon the extrapolated data, that high dose patients who respond to thecomposition of the invention, could lose up to about 10% of their totalbody weight in one year. Low dose patients could similarly lose about 6to about 7% of their total body weight.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A method of affecting weight loss in a patient inneed thereof, said method comprising administering to said patient aneffective amount of an orally bioavailable composition comprisinggelatin and constituents, wherein said constituents consist essentiallyof about 61 mole % 1,2 distearoyl-sn-glycero-3-phosphocholine, about 22mole % dihexadecyl phosphate, about 16 mole % cholesterol, and about 1mole % of a biotin-derived targeting agent, and wherein administrationof said gelatin and constituents to said patient affects weight loss insaid patient.
 2. The method of claim 1, wherein said biotin-derivedtargeting agent is D-biotin, biotin DHPE, or biotin-X-DHPE.
 3. Themethod of claim 1, further comprising co-administering to said patientat least one additional therapeutic agent useful for inducing weightloss.
 4. The method of claim 3, wherein the at least one additionaltherapeutic agent useful for inducing weight loss is orlistat,sibutramine, phendimetrazine tartrate, methamphetamine, IONAMIN™,phentermine, fenfluramine, dexfenfluramine, chitosan, chromiumpicolinate, conjugated linoleic acid, green tea extract, guar gum,hoodia, a combination of topiramate and phentermine, a combination ofbupropion and zonisamide, a combination of bupropion and naltrexone, acombination of phentermine and fluoxetine, a combination of phentermineand sertraline, a combination of phentermine and citalopram, acombination of phentermine and escitalopram, or a combination ofphentermine and trazodone.
 5. A method of maintaining the weight of apatient in need thereof, said method comprising administering to saidpatient a composition comprising gelatin and constituents, wherein saidconstituents consist essentially of about 61 mole % 1,2distearoyl-sn-glycero-3-phosphocholine, about 22 mole % dihexadecylphosphate, about 16 mole % cholesterol, and about 1 mole % of abiotin-derived targeting agent, and wherein administration of saidgelatin and constituents to said patient maintains said weight of saidpatient.
 6. The method of claim 5, wherein said biotin-derived targetingagent is selected from the group consisting of N-hydroxysuccinimide(NHS) biotin; sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin;sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin;sulfo-N-hydroxysuccinimide-S—S-biotin; biotin-BMCC; biotin-HPDP;iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytinhydrazide; biotin cadaverine; carboxybiotin; photobiotin; p-aminobenzoylbiocytin trifluoroacetate; p-diazobenzoyl biocytin; biotin DHPE;biotin-X-DHPE; 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide; biotinethylenediamine;biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE;biotin-XX, SSE; biotin-X-cadaverine; a-(t-BOC)biocytin;N-(biotinyl)-N′-(iodoacetyl)ethylenediamine; DNP-X-biocytin-X-SE;biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-l-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol)amine; (+)biotin 4-amidobenzoic acid sodium salt; Biotin2-N-acetylamino-2-deoxy-p-D-glucopyranoside;Biotin-a-D-N-acetylneuraminide; Biotin-a-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-a-D-mannopyranoside; biotin6-O-phospho-a-D-mannopyranoside; and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl),iminobiotin derivatives of the aforementioned compounds, and mixturesthereof.
 7. The method of claim 6, wherein said biotin-derived targetingagent is D-biotin, biotin DUPE, or biotin-X-DHPE.